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1. Validation of a high-confidence regulatory network for gene-to-NUE phenotype in field-grown rice

   https://doi.org/10.3389/fpls.2022.1006044  

   Shanks, Carly M.; Huang, Ji; Cheng, Chia-Yi; Shih, Hung-Jui S.; Brooks, Matthew D.; Alvarez, José M.; Araus, Viviana; Swift, Joseph; Henry, Amelia; Coruzzi, Gloria M. (November 2022, Frontiers in Plant Science)

   Nitrogen (N) and Water (W) - two resources critical for crop productivity – are becoming increasingly limited in soils globally. To address this issue, we aim to uncover the gene regulatory networks (GRNs) that regulate nitrogen use efficiency (NUE) - as a function of water availability - in Oryza sativa, a staple for 3.5 billion people. In this study, we infer and validate GRNs that correlate with rice NUE phenotypes affected by N-by-W availability in the field. We did this by exploiting RNA-seq and crop phenotype data from 19 rice varieties grown in a 2x2 N-by-W matrix in the field. First, to identify gene-to-NUE field phenotypes, we analyzed these datasets using weighted gene co-expression network analysis (WGCNA). This identified two network modules ("skyblue" & "grey60") highly correlated with NUE grain yield (NUEg). Next, we focused on 90 TFs contained in these two NUEg modules and predicted their genome-wide targets using the N-and/or-W response datasets using a random forest network inference approach (GENIE3). Next, to validate the GENIE3 TF→target gene predictions, we performed Precision/Recall Analysis (AUPR) using nine datasets for three TFs validated in planta . This analysis sets a precision threshold of 0.31, used to "prune" the GENIE3 network for high-confidence TF→target gene edges, comprising 88 TFs and 5,716 N-and/or-W response genes. Next, we ranked these 88 TFs based on their significant influence on NUEg target genes responsive to N and/or W signaling. This resulted in a list of 18 prioritized TFs that regulate 551 NUEg target genes responsive to N and/or W signals. We validated the direct regulated targets of two of these candidate NUEg TFs in a plant cell-based TF assay called TARGET, for which we also had in planta data for comparison. Gene ontology analysis revealed that 6/18 NUEg TFs - OsbZIP23 (LOC_Os02g52780), Oshox22 (LOC_Os04g45810), LOB39 (LOC_Os03g41330), Oshox13 (LOC_Os03g08960), LOC_Os11g38870, and LOC_Os06g14670 - regulate genes annotated for N and/or W signaling. Our results show that OsbZIP23 and Oshox22, known regulators of drought tolerance, also coordinate W-responses with NUEg. This validated network can aid in developing/breeding rice with improved yield on marginal, low N-input, drought-prone soils. 

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2. Systematic Discovery of Archaeal Transcription Factor Functions in Regulatory Networks through Quantitative Phenotyping Analysis

   https://doi.org/10.1128/mSystems.00032-17  

   Darnell, Cynthia L.; Tonner, Peter D.; Gulli, Jordan G.; Schmidler, Scott C.; Schmid, Amy K.; Shank, Elizabeth A. (September 2017, mSystems)

   ABSTRACT Gene regulatory networks (GRNs) are critical for dynamic transcriptional responses to environmental stress. However, the mechanisms by which GRN regulation adjusts physiology to enable stress survival remain unclear. Here we investigate the functions of transcription factors (TFs) within the global GRN of the stress-tolerant archaeal microorganism Halobacterium salinarum . We measured growth phenotypes of a panel of TF deletion mutants in high temporal resolution under heat shock, oxidative stress, and low-salinity conditions. To quantitate the noncanonical functional forms of the growth trajectories observed for these mutants, we developed a novel modeling framework based on Gaussian process regression and functional analysis of variance (FANOVA). We employ unique statistical tests to determine the significance of differential growth relative to the growth of the control strain. This analysis recapitulated known TF functions, revealed novel functions, and identified surprising secondary functions for characterized TFs. Strikingly, we observed that the majority of the TFs studied were required for growth under multiple stress conditions, pinpointing regulatory connections between the conditions tested. Correlations between quantitative phenotype trajectories of mutants are predictive of TF-TF connections within the GRN. These phenotypes are strongly concordant with predictions from statistical GRN models inferred from gene expression data alone. With genome-wide and targeted data sets, we provide detailed functional validation of novel TFs required for extreme oxidative stress and heat shock survival. Together, results presented in this study suggest that many TFs function under multiple conditions, thereby revealing high interconnectivity within the GRN and identifying the specific TFs required for communication between networks responding to disparate stressors. IMPORTANCE To ensure survival in the face of stress, microorganisms employ inducible damage repair pathways regulated by extensive and complex gene networks. Many archaea, microorganisms of the third domain of life, persist under extremes of temperature, salinity, and pH and under other conditions. In order to understand the cause-effect relationships between the dynamic function of the stress network and ultimate physiological consequences, this study characterized the physiological role of nearly one-third of all regulatory proteins known as transcription factors (TFs) in an archaeal organism. Using a unique quantitative phenotyping approach, we discovered functions for many novel TFs and revealed important secondary functions for known TFs. Surprisingly, many TFs are required for resisting multiple stressors, suggesting cross-regulation of stress responses. Through extensive validation experiments, we map the physiological roles of these novel TFs in stress response back to their position in the regulatory network wiring. This study advances understanding of the mechanisms underlying how microorganisms resist extreme stress. Given the generality of the methods employed, we expect that this study will enable future studies on how regulatory networks adjust cellular physiology in a diversity of organisms. 

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3. The MADS-domain factor AGAMOUS-Like18 promotes somatic embryogenesis

   https://doi.org/10.1093/plphys/kiab553  

   Paul, Priyanka; Joshi, Sanjay; Tian, Ran; Diogo Junior, Rubens; Chakrabarti, Manohar; Perry, Sharyn E. (November 2021, Plant Physiology)

   Abstract AGAMOUS-Like 18 (AGL18) is a MADS domain transcription factor (TF) that is structurally related to AGL15. Here we show that, like AGL15, AGL18 can promote somatic embryogenesis (SE) when ectopically expressed in Arabidopsis (Arabidopsis thaliana). Based on loss-of-function mutants, AGL15 and AGL18 have redundant functions in developmental processes such as SE. To understand the nature of this redundancy, we undertook a number of studies to look at the interaction between these factors. We studied the genome-wide direct targets of AGL18 to characterize its roles at the molecular level using chromatin immunoprecipitation (ChIP)-SEQ combined with RNA-SEQ. The results demonstrated that AGL18 binds to thousands of sites in the genome. Comparison of ChIP-SEQ data for AGL15 and AGL18 revealed substantial numbers of genes bound by both AGL15 and AGL18, but there were also differences. Gene ontology analysis revealed that target genes were enriched for seed, embryo, and reproductive development as well as hormone and stress responses. The results also demonstrated that AGL15 and AGL18 interact in a complex regulatory loop, where AGL15 inhibited transcript accumulation of AGL18, while AGL18 increased AGL15 transcript accumulation. Co-immunoprecipitation revealed an interaction between AGL18 and AGL15 in somatic embryo tissue. The binding and expression analyses revealed a complex crosstalk and interactions among embryo TFs and their target genes. In addition, our study also revealed that phosphorylation of AGL18 and AGL15 was crucial for the promotion of SE. 

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4. A new tool for discovering transcriptional regulators of co-expressed genes predicts gene regulatory networks that mediate ethylene-controlled root development

   https://doi.org/10.1093/insilicoplants/diaa006  

   Harkey, Alexandria F; Sims, Kira N; Muday, Gloria K (January 2020, in silico Plants)

   Marshall-Colon, Amy (Ed.)

   Abstract Gene regulatory networks (GRNs) are defined by a cascade of transcriptional events by which signals, such as hormones or environmental cues, change development. To understand these networks, it is necessary to link specific transcription factors (TFs) to the downstream gene targets whose expression they regulate. Although multiple methods provide information on the targets of a single TF, moving from groups of co-expressed genes to the TF that controls them is more difficult. To facilitate this bottom-up approach, we have developed a web application named TF DEACoN. This application uses a publicly available Arabidopsis thaliana DNA Affinity Purification (DAP-Seq) data set to search for TFs that show enriched binding to groups of co-regulated genes. We used TF DEACoN to examine groups of transcripts regulated by treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), using a transcriptional data set performed with high temporal resolution. We demonstrate the utility of this application when co-regulated genes are divided by timing of response or cell-type-specific information, which provides more information on TF/target relationships than when less defined and larger groups of co-regulated genes are used. This approach predicted TFs that may participate in ethylene-modulated root development including the TF NAM (NO APICAL MERISTEM). We used a genetic approach to show that a mutation in NAM reduces the negative regulation of lateral root development by ACC. The combination of filtering and TF DEACoN used here can be applied to any group of co-regulated genes to predict GRNs that control coordinated transcriptional responses. 

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5. Rapid rewiring of an archaeal transcription factor function via flexible cis–trans interactions

   https://doi.org/10.1091/mbc.E24-11-0505  

   Pastor, Mar Martinez; Darnell, Cynthia L; Vreugdenhil, Angie; Schmid, Amy K (July 2025, Molecular Biology of the Cell)

   Garner, Ethan (Ed.)

   For microbial cells, an appropriate response to changing environmental conditions is critical for viability. Transcription regulatory proteins, or transcription factors (TF) sense environmental signals to change gene expression. However, it remains unclear how TFs and their corresponding gene regulatory networks are selected over evolutionary time scales. The function of TFs and how they evolve are particularly understudied in archaeal organisms. Here, we identified, characterized, and compared the function of the RosR TF across three related hypersaline-adapted archaeal model species. RosR was previously characterized as a global regulator of gene expression during oxidative stress in the species Halobacterium salinarum ( hsRosR). Here, we use functional genomics and quantitative phenotyping to demonstrate that, despite strong sequence conservation of RosR across species, its function diverges substantially. Surprisingly, RosR in Haloferax volcanii ( hvRosR) and Haloferax mediterranei ( hmRosR) regulates genes whose products function in motility and the membrane, leading to significant defects in motility when RosR is deleted. Given weak conservation and degeneration in cis-regulatory sequences recognized by the RosR TF across species, we hypothesize that the RosR regulatory network is readily rewired during evolution across related species of archaea. 

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